The present invention relates to instruments and methods for the temperature dependent analysis of nucleic acids, peptides, carbohydrates, and the like (hereinafter biological oligomers) and in particular to an instrument that can assist in quantitative analysis of binding interactions of such oligomers.
Measuring the thermodynamics of interactions involving nucleic acids and peptides is a vital component of biomolecular studies. Conventionally, these properties are analyzed in solution by temperature dependent spectroscopic measurement (e.g., UV-vis, CD, fluorescence) or thermal methods (ITC, DSC). See generally (1) Mergny, J. L.; Lacroix, L. Oligonucleotides 2003, 13, 515-537 and Jelesarov, I.; Bosshard. H. R. J. Mol. Recognit. 1999, 12, 3-18. For example, DNA thermal denaturation profiles obtained by monitoring changes in UV absorption as the temperature of the solution is incremented can be used to generate robust models for predicting the stabilities of DNA structures in solutions.
Surface-based diagnostics, such as surface plasmon resonance (SPR), have been used to provide analysis of binding kinetics and affinities of biomolecular interactions at a single temperature. In SPR, a nanoscale thin metallic film is illuminated from the back “reflecting side” of the film. At a certain angle, known as the plasmon angle, the energy from the illumination is coupled into electromagnetic waves creating a resonant condition (surface plasmon resonance) that is highly sensitive to surface conditions on the “sensing side” of the film opposite to the reflecting side. To the extent that SPR can directly detect linking between probe molecules on the film and target molecules in a solution, largely eliminates the need to use fluorescent or enzymatic tags for monitoring such linking.
Specifically, in SPR imaging, multiple probe spots of a micro-array may be analyzed in parallel, with different biological oligomer probes attached over the surface. In this technique of SPR imaging, p-polarized light impinges on a prism, a gold thin film, and a flow cell assembly at a fixed angle. The reflected light is passed through a narrow band pass filter and collected by a CCD camera. In this technique, probe molecules are covalently linked to discrete positions on the sensing side of the film to selectively bind with target molecules in the solution to be analyzed. The binding of the targets to the surface bound probes causes localized changes in the index of refraction at those spots, which are detected by the CCD camera. This data can then be used to identify the presence and composition of the target molecules by the location of the detected binding event.
SPR experiments can be repeated at different temperatures to provide a better understanding of interactions between biological oligomers, for example, indicating the temperatures at which binding interactions occur or providing plots showing how sensor binding kinetics varies with temperature. Because SPR measurements are highly sensitive to temperature variation, for each experiment the SPR apparatus is normally stabilized to a uniform temperature. Changing this uniform temperature between measurements is normally accompanied by an extended stabilization time during which the apparatus acclimates to a uniform and precise temperature greatly slowing the acquisition of data.